Cellular processes are highly sensitive to ionic conditions. A key element of control of intracellular ionic balance in mammalian cells is plasma membrane Na/H exchangers (NHE), which as such indirectly control many physiological processes such as cell volumes cell proliferation and transcellular Na absorption. There are 4 known Na/H exchanger isoforms in eukaryotic cells, which are named in order of their cloning as NHE1, NHE2, NHE3, NHE4. The first 3 have been shown to function as Na/H exchangers when expressed in cells deficient in endogenous Na/H exchangers. NHE1 and NHE3 are regulated by protein kinases and growth factors via different kinetics. NHE1 is stimulated via increased sensitivity to intracellular H (Km change) while NHE3 is regulated by changes in Vmax. Detailed biochemical studies performed on NHE1 and preliminary studies with NHE3 have shown that calmodulin is a regulator of both isoforms, stimulating NHE1 and inhibiting NHE3. The aims of this project are to 1) determine which part of the NHE N- terminus molecule explains the Km vs Vmax regulation and 2) identify mechanisms by which calmodulin regulates NHE3. Point mutation, PCR, ligation, transfection, calmodulin affinity chromatography will be used.